This project encompasses a number of separate research problems relevant to our understanding of molecular mechanisms for replication and transcription. Research effort currently includes examination of the following problems: (a) Replication of the terminal segment in the E.coli chromosome. Our studies on the effect of temperature shifts on the incorporation of labeled thymidine into DNA in E. coli do not support the hypothesis that a unique terminal segment exists and that protein synthesis is required for the synthesis of that segment. We are now examining the effect of temperature shifts on rates of DNA replication. The size and specific activity of the thymidine triphosphate pool is being studied using a two-dimensional thin layer chromatography system. Attempts are also being made to develop a one-dimensional system suitable for use with P32-labeled cell extracts. (b) Intermediates in DNA replication in P4 bacteriophage: Studies on the size distribution and classes of intermediates in the replication of this small genome are being carried out to determine whether Okazaki fragments are uniquely specified. (c) Thymineless death and DNA "turnover" in bacteria. We are testing our hypothesis that thymineless death (tld) results from the accumulation of unrepaired single-strand break in DNA. Similarities between tld and the lethality of dnaF strains (defective in ribonucleoside reductase) are being examined. Our hypothesis that transcription introduces repairable single strand breaks in bacterial DNA is being tested by selective inhibition of transcription in RNA polymerase deficient mutants or by rifampin, puromycin, or chloramphenicol treatment and subsequent examination of DNA molecular weight distribution by alkaline sucrose gradient sedimentation. (d) Effects of 5-bromouracil in DNA. The pathogenic effects of 5-bromouracil in bacteria and in phage lambda are being studied in parallel with an examination of abnormalities in replication and recombination when the analogue is substituted for thymine. BIBLIOGRAPHIC REFERENCES: Hanawalt, P. "Molecular mechanisms involved in DNA repair" Genetics 79:Symposia Supplement, Part II, pp. 179-197 (1975).